Summary NMHC IIA was immunoprecipitated in multi-protein complexes from HeLa cell lysates with antibodies against BIG1 or BIG2 and identified via liquid chromatography MS/MS analysis of tryptic peptides from the collected proteins. To assess potential interactions of NMHC IIA with BIG1 and/or BIG2, we analyzed proteins co-immunoprecipitated from HeLa cells with antibodies against BIG1 or BIG2. IP of endogenous NMHC IIA yielded also BIG1 and BIG2. BIG1 and BIG2 interactions with NMHC IIA were further explored after selective depletion of BIG1 or BIG2 with siRNA. Although BIG2 depletion did not alter IP of NMHC IIA by BIG1 antibodies, IP of BIG2 after BIG1 depletion yielded about twice as much NMHC IIA, suggesting BIG1 priority in competition of the endogenous proteins for interaction with NMHC IIA. IP of NMHC IIA similarly yielded more than twice as much BIG2 from BIG1-depleted as from control (NT) cells. To identify regions of the BIG1 molecule that interacted with NMHC IIA, HeLa cells were co-transfected with constructs encoding BIG1 amino acids 1-1849 (F, full-length), 1-885 (N), 698-885 (S), or 886-1849 (C) with N-terminal HA tags and GFP-NMHC IIA (full-length) 24 h before preparation of lysates for IP with antibodies against GFP or HA. Data were consistent with the conclusion that NMHC IIA interacted with structural elements in the C-terminal region of BIG1. Reversible phosphorylation of RLC T18 and S19 determines MN IIA activity. After BIG1 or BIG2 depletion, phosphorylation of RLC T18 and S19 was more than twice that in cells treated with the non-targeted (NT) siRNA or vehicle alone. Distributions of phosphorylated RLC in BIG1- or BIG2-depleted and control cells were compared microscopically. BIG1 or BIG2 siRNA treatment increased significantly the mean fluorescence intensity of phospho-RLC (T18/S19). Linear arrangements of phospho-RLC that co-localized with F-actin were clear in BIG1- or BIG2-depleted cells. As NM II activity is known to be related to F-actin organization, we looked for effects of BIG1 or BIG2 depletion on F-actin morphology. Stress fibers were significantly more prominent after BIG1 or BIG2 depletion than in control cells, consistent with the effects of BIG1 or BIG2 on RLC phosphorylation. We did not observe an association of BIG1 and BIG2 with three known NM II kinases, ROCK1, ROCK2 and MLCK. Endogenous protein phosphatase 1 (PP1), reported to be responsible for dephosphorylation of myosin IIA, was among proteins collected by BIG1 and/or BIG2 IP. More notably, however, co-IP of PP1 with NMHC IIA was significantly decreased after BIG1 or BIG2 depletion, consistent with BIG1 and BIG2 influences via PP1 on levels of RLC phosphorylation. Mammalian cells contain several forms of PP1c. Association of different PP1c forms with BIG1 or BIG2 in HeLa cells was compared and PP1c-delta; was among proteins precipitated with BIG1 or BIG2 antibodies. Consistent with the conclusion that PP1c-delta; was the catalytic subunit of the NM II phosphatase, IP with PP1c-delta; antibodies yielded also BIG1 and BIG2., co-IP of PP1c-delta; with NMHC IIA after BIG1 or BIG2 depletion was significantly less than that after treatment with control siRNA. Myosin phosphatase is a heterotrimer comprising the catalytic subunit of protein phosphatase type 1c-delta, plus the 130-kDa myosin-binding MYPT1 (myosin phosphatase-targeting subunit 1) and a smaller subunit (M20) of unknown function. Phosphatase activity of the holoenzyme was reported to be greater than that of the free catalytic subunit, suggesting that myosin-binding by MYPT1 facilitated enzyme-substrate interaction. Thus, BIG1 and BIG2 could influence an NMHC IIA-PP1c-delta interaction, perhaps involving MYPT1. To test this possibility, we looked for and found endogenous MYPT1 among proteins precipitated with antibodies against BIG1 or BIG2, but not control IgG. Reciprocal IP with antibodies against MYPT1 collected also BIG1 and BIG2. Co-IP of MYPT1 with NMHC IIA was ca. 50% lower after BIG1 or BIG2 depletion, consistent with their participation in assembly and/or stabilization of the large molecular complexes. Phosphorylations of NM II RLC T18 and S19 enhance assembly and activity of NM II. To explore whether levels of RLC phosphorylation affected amounts of these multimolecular complexes, three RLC constructs with C-terminal GFP tags, wild type RLC, non-phosphorylatable (RLC-T18A/S19A), or phosphomimetic (RLC-T18D/S19D), were overexpressed in HeLa cells. Cells with RLC-T18D/S19D appeared to have more NM IIA and RLC in stress fiber-like arrangements than did those containing wild type RLC or RLC-T18A/S19A. Associations of BIG1 and BIG2 with NM IIA, PP1c-delta;, and MYPT1 increased as the amount of overexpressed RLC-T18D/S19D, but not RLC-T18A/S19A, increased, consistent with a dependence on the negative charge conferred by phosphorylation of T18/S19. All of our data suggested the existence of endogenous BIG1, BIG2, NMHC IIA, MYPT1, PP1c-delta;, and perhaps additional proteins in macromolecular complexes. To evaluate possibly direct interactions of individual components, proteins synthesized in vitro (wheat germ extract) were used. About 2% of BIG1-F and -C were found after IP of NMHC IIA. Direct interactions of MYPT1 with NM IIA and PP1c-delta had been reported. An absence of evidence that depletion of NM IIA affected coIP of endogenous MYPT1 and PP1c-delta with BIG1 was consistent with direct interactions of BIG1 and those two proteins, which was confirmed by co-IP of ca. 2% of in vitro-synthesized HA-BIG1-C with MYPT1 and PP1c-delta. Despite > 70% sequence identity of the two C fragments, no interaction of BIG2-C with MYPT1 or PP1c-delta was detected. BIG2 appeared to interact with BIG1 and NMHC IIA. As NM IIA is critical in cell motility, roles of BIG1 and BIG2 in cell movement were evaluated by Transwell migration assays, and findings were consistent with reports that depletion of BIG1 or BIG2 interfered with cell migration, albeit in different ways. Motility of HeLa cells transfected with BIG1 or BIG2 siRNA was significantly impaired relative to that of cells transfected with NT siRNA, and overexpression of BIG1 or BIG2, full length or C fragment, but not N or S fragments, reversed those effects. Effects on stress fibers were similar, perhaps reflecting the changes in RLC phosphorylation. Reversal of effects of BIG1 or BIG2 depletion on co-IP of PP1&#948; and MYPT1 with NMHC IIA was associated with dephosphorylation of RLC. As C fragments effectively replaced the full-length proteins in these experiments, a dependence on Arf GEF activity was excluded. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1 delta, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells.